CN112055717A - Neutralization of human cytokines with membrane-bound anti-cytokine non-signaling adhesive expressed in immune cells - Google Patents

Abstract

Transgenic T cells and vectors for making transgenic T cells are described. These vectors may include nucleic acids encoding membrane-bound anti-IL 6(mb-aIL6) single-chain variable fragments (scFv), and these transgenic T cells may express mb-aIL 6. These transgenic T cells can be used to inhibit proliferation of IL-6 dependent cells, to reduce IL-6 concentration, or both. In one embodiment, the vector is a bicistronic construct encoding the mb-alL6 and anti-CD 19-41 BB-003 zeta Chimeric Antigen Receptor (CAR). In another embodiment, the anti-IL-6 scFv can be linked to the 41BB and 003 zeta domains to form an anti-IL-6 CAR. Transgenic T cells expressing the constructs can reduce the risk of Cytokine Release Syndrome (CRS) in cancer patients undergoing CAR T cell therapy, or for the treatment of autoimmune and inflammatory diseases where cytokines are involved in pathogenesis.

Description

Neutralization of human cytokines with membrane-bound anti-cytokine non-signaling adhesive expressed in immune cells

Cross Reference to Related Applications

This application claims the benefit of U.S. provisional application No. 62/651,311 filed on 2.4.2018. The entire teachings of the above application are incorporated herein by reference.

The material in the ASCII text file is incorporated by reference

The present application incorporates by reference the sequence listing contained in the following ASCII text file, which is filed concurrently herewith:

a) file name: 44591149001sequence listing.txt; created on day 29 of month 3 2019, 33KB in size.

Background

Interleukin-6 (IL-6) is a proinflammatory cytokine involved in the pathogenesis of a variety of autoimmune and inflammatory diseases, including rheumatoid arthritis, systemic lupus erythematosus, and graft-versus-host disease (GvHD).

IL-6 is also involved in the development of Cytokine Release Syndrome (CRS), one of the most common side effects following infusion of T cells redirected with Chimeric Antigen Receptors (CARs). CRS can be severe and in some cases fatal. IL-6 is critical for the development of CRS and an anti-IL-6 receptor antibody (truzumab) is currently used to suppress its effects.

Methods for reducing the severity and incidence of autoimmune disease and CRS are desirable.

Disclosure of Invention

Nucleic acids, vectors, and transgenic host cells are described herein, as well as methods of making and using these nucleic acids, vectors, and transgenic host cells. The nucleic acid may be incorporated into a vector, which may be used to express the nucleic acid in a host cell. The transgenic host cell can be introduced (e.g., transfused, implanted, engrafted, injected) into a host mammal (e.g., a human) in a method of reducing the concentration of a cytokine (e.g., IL-6). Nucleic acids can be expressed in a host mammal (e.g., a human) in a method of reducing the concentration of a cytokine (e.g., IL-6).

In some embodiments, the vector comprises a nucleic acid encoding a membrane-bound anti-cytokine single-chain variable fragment (scFv). The membrane-bound anti-cytokine may include an anti-cytokine single-chain variable fragment (anti-cytokine scFv), and a hinge and transmembrane domain coupled to the anti-cytokine scFv. The anti-cytokine scFv may comprise an anti-cytokine variable light domain, an anti-cytokine variable heavy domain, and a linker domain connecting the variable light domain and the variable heavy domain. The anti-cytokine construct can be specific for a variety of cytokines, such as IL-6 (e.g., anti-IL-6), (TNF) - α (e.g., anti-TNF- α), IL-1 β (e.g., anti-IL-1 β), IL-12 (e.g., anti-IL-12), IL-17 (e.g., anti-IL-17), IL-18 (e.g., anti-IL-18), IFN γ (e.g., anti-IFN γ), and the like.

In some embodiments, the nucleic acid encodes a membrane-bound anti-IL 6(mb-aIL6) single-chain variable fragment (scFv). mb-aIL6 may include an anti-IL 6 single chain variable fragment (anti-IL 6 scFv). The anti-IL 6scFv may include an anti-IL-6 variable light domain, an anti-IL-6 variable heavy domain, and a linker domain that links the variable light domain and the variable heavy domain. The hinge and transmembrane domains may be conjugated to an anti-IL 6 scFv. In some embodiments, the nucleic acid of the vector can further encode a Chimeric Antigen Receptor (CAR), such as anti-CD 19-41BB-CD3 ζ.

The vectors described herein can be used to generate transgenic cells, such as transgenic T cells. In particular, the transgenic T cells can be used to inhibit proliferation of IL-6 dependent cells, reduce IL-6 concentration, or both. In some embodiments, the transgenic T cells can be used to reduce the risk or severity of Cytokine Release Syndrome (CRS) in a mammal (e.g., a human), such as a mammal being treated (e.g., for cancer). In some embodiments, the mammal is receiving treatment with a Chimeric Antigen Receptor (CAR) T cell (e.g., treatment for cancer). One example is that the patient is receiving treatment for cancer with T cells expressing an anti-CD 19 CAR.

The transgenic T cells can also be used to treat a mammal having a disease or disorder in which a cytokine is involved in pathogenesis, such as an autoimmune disease, an inflammatory disease, or a lymphoproliferative disorder. Examples of autoimmune diseases include rheumatoid arthritis and systemic lupus erythematosus. Examples of inflammatory diseases include graft versus host disease and hemophagocytic lymphohistiocytosis. An example of a lymphoproliferative disorder is Castleman disease (Castleman disease).

Drawings

The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The figures are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

FIGS. 1A-D relate to the design and expression of mb-aIL 6. FIG. 1A is a schematic representation of the mb-aIL6 construct. FIG. 1B is a schematic representation of the MSCV-mb-aIL6-IRES-GFP plasmid. FIG. 1C is a flow cytometric analysis of Jurkat cells transduced with GFP alone ("mock") or GFP plus mb-aIL 6. Dot plots show GFP fluorescence and mb-aIL6 expression after staining with biotin conjugated goat anti-human F (ab') 2 antibody and streptavidin APC (Jackson ImmunoResearch Laboratories). FIG. 1D is a flow cytometric analysis of IL-6 binding to mock-transduced or mb-aIL6 transduced Jurkat cells. Dot plots show the binding of IL-6 to GFP fluorescence after staining with IL-6 biotin (Abcam) and streptavidin APC. Soybean Trypsin Inhibitor (STI) -biotin was used as a control.

FIGS. 2A-E relate to the function of the mb-aIL6 construct. FIG. 2A is a graph showing the level of IL-6. Jurkat cells transduced with GFP alone ("mock") or GFP plus mb-aIL6(2x10⁶/mL) was cultured in tissue culture medium containing 1ng/mL human IL-6 for 2 hours. At the end of the incubation, the level of IL-6 was measured by ELISA. FIG. 2B is a graph showing that IL-6 neutralization is cell dose-dependent. Different cell concentrations (0.25-2X 10) were tested⁶mL), and the level of IL-6 in the supernatant after 2 hours of culture was measured by ELISA. FIG. 2C is a graph showing that IL-6 neutralization is time-dependent. Cultures were established as described for figure 2A, and the level of IL-6 in the supernatant was measured after the indicated incubation time. Cells were incubated for 10, 30, 60 and 120 minutes. The dashed curve represents the fitted exponential decay curve. FIG. 2D is a graph showing that mb-aIL6 Jurkat cells were still effective at low IL-6 concentrations. Cultures were established as described with respect to FIG. 2A, but the initial concentration of IL-6 in the tissue culture medium ranged from 0.025 to 0.2ng/mL and the initial concentration of Jurkat cells was 0.5X10⁶and/mL. FIG. 2E is a graph showing that IL-6 neutralization was enhanced by allowing Jurkat-mb-aIL6 cells to proliferate. Cultures were established as described with respect to fig. 2A, but with an initial cell concentration of 0.2x10⁶Individual cells/mL; IL-6 in the supernatant was measured by ELISA after 2-72 hours.

FIGS. 3A-B show that cells expressing mb-aIL6 abolished IL-6-dependent signaling and cell proliferation. FIG. 3A is a graph showing the prevention of Stat3 phosphorylation in U937 cells triggered by IL-6 by exposure to mb-aIL6 Jurkat cells. Jurkat cells (2X 10) untransduced ("WT") or transduced with GFP plus mb-aIL6⁶/mL) was cultured in tissue culture medium containing 1ng/mL human IL-6 for 2 hours. The supernatant was then added to U937 cells at 37 ℃ for 15 minutes. Tissue culture medium with or without IL-6 ("T cell free") was used as a control. The histogram shows the mean (± SD) of Stat3 phosphorylation as measured by flow cytometry after staining with anti-phosphorylated Stat3 antibody (BD Biosciences anti-Stat 3 pY 705). P<0.001. FIG. 3B is a graph showing inhibition of IL-6-dependent proliferation of DS-1 cells by exposure to mb-aIL6 Jurkat cells. DS-1 and mock-transduced or mb-IL 6-transduced Jurkat cells transduced with mCherry were co-cultured with IL-6(0.5ng/mL) at a ratio of 1: 1. Quantification of DS-1 proliferation using IncuCyte biopsy system (Essen);results are expressed as Red Calibration Units (RCU) x μm of triplicate measurements²Mean value (± SD)/well. P<0.01 for a measurement of 120 hours.

FIGS. 4A-D show expression and function of mb-aIL6 constructs on peripheral blood T cells. FIG. 4A is a flow cytometric analysis of peripheral blood T cells transduced with GFP alone ("mock") or GFP plus mb-aIL 6. Dot plots show GFP fluorescence and mb-aIL6 expression after staining with biotin-conjugated goat anti-human F (ab') 2 antibody and streptavidin APC. FIG. 4B is a flow cytometric analysis of IL-6 binding to either mock-transduced or mb-aIL 6-transduced peripheral blood T cells. Dot plots show GFP fluorescence and IL-6 binding after staining with IL-6 biotin and streptavidin APC. FIG. 4C is a cell signature profile of peripheral blood T cells labeled with anti-CD 3 APC, anti-CD 56 PE, anti-CD 4V 450, and anti-CD 8 PerCP (BD biosciences), mock-transduced, or mb-aIL6 transduced. FIG. 4D is a graph showing inhibition of IL-6-dependent proliferation of DS-1 cells by exposure to mb-aIL 6T lymphocytes. DS-1 and mock-transduced or mb-aIL 6-transduced T cells transduced with mCherry were co-cultured with IL-6(0.5ng/mL) at a ratio of 1: 1. Quantification of DS-1 proliferation using IncuCyte in vivo imaging System (Itson corporation); results are expressed as Red Calibration Units (RCU) x μm of triplicate measurements²Mean value (± SD)/well. P<0.01 for a measurement of 120 hours.

FIGS. 5A-C show the design, expression, and IL-6 neutralizing ability of bicistronic constructs encoding mb-aIL6 and an anti-CD 19 CAR. FIG. 5A is a schematic representation of the MSCV plasmid containing two receptors ("DUAL"). FIG. 5B is a flow cytometric analysis of peripheral blood T cells transduced with GFP alone ("mock"), GFP plus anti-CD 19-41BB-CD3 ζ CAR, mb-aIL6, or both. Dot plots show mb-aIL6 expression after staining with biotin-conjugated goat anti-human F (ab') 2 antibody and streptavidin APC, and anti-CD 19CAR expression after staining with CD19-myc followed by R-Phycoerythrin (PE) -conjugated anti-myc (Cell Signaling Technology). Figure 5C is a graph showing that mb-aIL 6T lymphocytes and IL-6 were not affected by CAR co-expression. T lymphocytes transduced as described for FIG. 5B were cultured in tissue culture medium containing 1ng/mL IL-6. After 2 hours, IL-6 in the supernatant was measured by ELISA.

FIGS. 6A-D show that expression of mb-aIL6 did not affect anti-CD 19CAR function. FIG. 6A is a graph showing IFN- γ production in T cells co-cultured with CD19+ ALL cell line OP-1 for 6 hours at an E: T ratio of 1: 1. After staining with PE-conjugated anti-human IFN γ antibody (BD biosciences), the expression of IFN γ was measured by flow cytometry. Symbols represent the results of triplicate experiments obtained with T cells from 3 donors. FIG. 6B is a graph showing CD107a expression in T cells co-cultured with OP-1 for 4 hours at an E: T ratio of 1: 1. CD107a expression was measured by flow cytometry after staining with PE-conjugated anti-human CD107a antibody (BD biosciences). FIG. 6C is a graph showing cytotoxicity of T cells to OP-1 after 4 hours at an E: T ratio of 1: 1. FIG. 6D is two graphs showing the proliferation of T cells cultured at an E: T ratio of 1:1 with or without irradiated OP-1 (containing 120IU/mL IL-2) for 21 days. Irradiated OP-1 cells were added on days 0, 7 and 14. Symbols represent the mean (± SD) of triplicate measurements.

Figures 7A-B show the function of T cells expressing mb-aIL6 and anti-CD 19CAR in an in vitro model of CRS. FIG. 7A are two graphs showing cytotoxicity of T cells co-cultured with mCherry transduced OP-1 cells with or without THP-1 cells (1:5:1T cells: OP-1: THP-1 ratio). Quantification of OP-1 cell number using IncuCyte in vivo imaging System (Itson corporation); results are expressed as Red Calibration Units (RCU) x μm of triplicate measurements²Mean value (± SD)/well. FIG. 7B is a graph showing the level of IL-6 in the supernatant of the culture shown in FIG. 7A measured by ELISA after 40 hours of culture.

FIGS. 8A-C are schematic representations of other IL-6 neutralizing receptors. Figure 8A is a schematic of a nucleic acid construct directed to secreted aIL6 and a cell expressing secreted aIL 6. Figure 8B is a schematic of a nucleic acid construct directed against an anti-IL-6 scFv linked to a 41BB domain and a CD3 zeta domain, thereby forming an anti-IL 6 CAR. FIG. 8C is a schematic representation of a nucleic acid construct in which the anti-IL 6scFv was replaced with an IL-6 receptor that lost signaling ability.

FIGS. 9A-B show mb in T cellsExpression of-aIL 6 neutralizes IL-6 in vivo. Cg-Prkdc in day 0^scid IL2rg^tm1WjlPer P.I. injection of 1X 10/SzJ (NOD/scid IL2RGnull) mice (Jackson Laboratory, Balkong, Maine)⁶DS-1 cells and injected intraperitoneally with 1x10 transduced with GFP alone ("mock") or GFP plus mb-aIL6 on day 2⁷And (3) peripheral blood T cells. Tumor implantation and growth were measured using the Xenogen IVIS-200 system (Caliper Life Sciences). Starting on day 0, all mice received 1000IU human IL-6 and 20000IU human IL-2 intraperitoneally every 2 days. Fig. 9A is a ventral and dorsal image of the mouse. FIG. 9B is a graph showing the change in luminescence in mice implanted with luciferase-expressing DS-1. Each symbol corresponds to a bioluminescence measurement; the line connects all measurements in one mouse.

FIGS. 10A-F show that expression of mb-aIL6 did not affect anti-CD 19CAR function in vivo. Cg-Prkdc in day 0^scid IL2rg^tm1Wjl/SzJ (NOD/scid IL2RGnull) mice (Jackson laboratory, Balanus, Myain) were injected intravenously with 0.5x10⁶Nalm-6 cells and injected intravenously on day 3 with 2x10 transduced with anti-CD 19-41BB-CD3 ζ (CAR) or CAR plus mb-aIL6(CAR + mb-aIL6)⁷And (3) peripheral blood T cells. Tumor implantation and growth were measured using the Xenogen IVIS-200 system (caliper life sciences). Starting on day 0, every 2 days all mice received 20000IU human IL-2 intraperitoneally. When the signal threshold reaches 10¹⁰Mice were euthanized at one photon/second. Fig. 10A is ventral and dorsal images of mice. Day 3 images were processed with enhanced sensitivity to show the presence of tumors prior to injection of engineered T cells. FIG. 10B is a graph showing the change in luminescence in mice implanted with luciferase-expressing Nalm-6. Fig. 10C is ventral and dorsal images of mice. Day 3 images were processed with enhanced sensitivity to show the presence of tumors prior to injection of engineered T cells. Figure 10D is a graph showing CAR T cell counts at day 53. Mouse blood was obtained via buchner puncture and CAR T cells were quantified using flow cytometry. Fig. 10E is a graph showing survival curves of mice. By logarithmRank test the curve was calculated for mice not injected with T cells versus mice injected with CAR-T cells or CAR-T + mb-aIL6 (P)<0.01 for either comparison). FIG. 10F is a graph showing the change in luminescence in mice implanted with luciferase-expressing Nalm-6. Each symbol corresponds to a bioluminescence measurement; the line connects all measurements in one mouse.

FIGS. 11A-B relate to the design of mb-aTNF α. FIG. 11A is a schematic representation of the mb-aTNF α construct. FIG. 11B is a schematic representation of the MSCV-mb-aTNF α -IRES-GFP plasmid.

Detailed Description

The description of the example embodiments follows.

Interleukin-6 and cytokine release syndrome

Interleukin-6 (IL-6) is a proinflammatory cytokine involved in the pathogenesis of a variety of autoimmune diseases, inflammatory diseases, and lymphoproliferative disorders, including graft versus host disease (GvHD), rheumatoid arthritis, and systemic lupus erythematosus. IL-6 is also involved in the development of Cytokine Release Syndrome (CRS), one of the most common side effects following infusion of T cells redirected with Chimeric Antigen Receptors (CARs). CRS can be severe and in some cases fatal.^7-10IL-6 is critical for the development of CRS and an anti-IL-6 receptor antibody (truzumab) is currently used to suppress its effects.^7-10

The vectors described herein are useful for generating modified T cells, which in turn are useful for treating autoimmune diseases and CRS. The processes described herein can be used to generate transgenic T cells that can neutralize IL-6, thereby reducing the risk and/or severity of CRS. Although the specific examples described herein use IL-6 as an example, the method is applicable to neutralize other cytokines involved in the pathogenesis of autoimmune diseases and CRS.

Nucleic acids

As used herein, the term "nucleic acid" refers to a polymer comprising a plurality of nucleotide monomers (e.g., ribonucleotide monomers or deoxyribonucleotide monomers). "nucleic acid" includes, for example, DNA (e.g., genomic DNA and cDNA), RNA, and DNA-RNA hybrid molecules. The nucleic acid molecule may be naturally occurring, recombinant or synthetic. In addition, the nucleic acid molecule may be single-stranded, double-stranded or triple-stranded. In certain embodiments, the nucleic acid molecule may be modified. In the case of a double-stranded polymer, "nucleic acid" may refer to either or both strands of the molecule.

The terms "nucleotide" and "nucleotide monomer" refer to naturally occurring ribonucleotide or deoxyribonucleotide monomers, as well as non-naturally occurring derivatives and analogs thereof. Accordingly, nucleotides can include, for example, nucleotides comprising naturally occurring bases (e.g., adenosine, thymidine, guanosine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine, or deoxycytidine) and nucleotides comprising modified bases known in the art.

As used herein, the term "sequence identity" refers to the degree to which two nucleotide sequences or two amino acid sequences have identical residues at the same position, expressed as a percentage, when the sequences are aligned to achieve the maximum level of identity. For sequence alignment and comparison, one sequence is typically designated as a reference sequence and compared to a test sequence. Sequence identity between a reference sequence and a test sequence is expressed as a percentage of positions across the entire length of the reference sequence, where the reference sequence and the test sequence share the same nucleotides or amino acids when aligned to achieve a maximum level of identity. As an example, when aligned to achieve a maximum level of identity, two sequences are considered to have 70% sequence identity, with the test sequence having the same nucleotide or amino acid residue at the same position for 70% of the entire length of the reference sequence.

One of ordinary skill in the art can readily perform sequence alignments for comparison using appropriate alignment methods or algorithms to achieve maximum levels of identity. In some cases, the alignment may include gaps introduced to provide a maximum level of identity. Examples include the homology alignment algorithms of Smith and Waterman, adv.Appl.Math. [ applied math progress ]2:482(1981), the homology alignment algorithms of Needleman and Wunsch, J.mol.biol. [ journal of Molecular Biology ]48:443(1970), the homology method studies of Pearson and Lipman, Proc.Natl.Acad.Sci.USA [ Proc. Natl.Acad.Sci.85: 2444 (1988)), the Computer implementation of these algorithms (GAP, BESTFIT, ASTA, and TFTA in the Wis. Concercosungs. Genet. of Madison, genetic Computer Group 575 (Genetics Computer Group,575Science Dr., Madison, Wis.), or the visual inspection (see general FASSUSURE et al, Current Molecular Biology laboratory).

When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of one or more test sequences relative to the reference sequence based on the specified program parameters. A common tool for determining percent sequence identity is the protein-Based Local Alignment Search Tool (BLASTP), which is available through the United States National Center for Biotechnology Information of the National Library of Medicine (National Library of Medicine) of the National Institutes of Health (United States State National Institutes of Health). (Altschul et al, J Mol Biol. [ J. Mobiol. ]215(3):403-10 (1990)).

In different embodiments, two nucleotide sequences or two amino acid sequences can have at least, e.g., 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity. When determining percent sequence identity to one or more sequences described herein, a sequence described herein is a reference sequence.

In some embodiments, the variable light chain domain has at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID No. 4. In some embodiments, the variable heavy chain domain has at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID No. 8.

Carrier

The terms "vector," "vector construct," and "expression vector" mean a vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, thereby transforming the host and promoting expression (e.g., transcription and translation) of the introduced sequence. Vectors typically comprise DNA capable of transmitting an agent into which foreign DNA encoding a protein has been inserted by restriction enzyme techniques. A common type of vector is a "plasmid", which is typically a self-contained (self-contained) molecule of double-stranded DNA that can readily accept other (foreign) DNA and can be readily introduced into a suitable host cell. A number of vectors, including plasmids and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts.

The term "expression" means allowing or allowing the expression of information in a gene or DNA sequence, for example the production of a protein by activating a cellular function related to the transcription and translation of the corresponding gene or DNA sequence. The DNA sequence is expressed in or by a cell to form an "expression product" such as a protein. The expression product itself, e.g., the resulting protein, may also be said to be "expressed" by the cell. For example, a polynucleotide or polypeptide is recombinantly expressed or produced in a foreign host cell under the control of a foreign or native promoter, or expressed or produced in a native host cell under the control of a foreign promoter.

Gene delivery vectors typically include a transgene (e.g., a nucleic acid encoding an enzyme) operably linked to a promoter and other nucleic acid elements required for expression of the transgene in a host cell into which the vector is introduced. Suitable promoters and delivery constructs for gene expression are known in the art. The recombinant plasmid may also contain an inducible or regulatable promoter for expression of the enzyme in the cell.

A variety of gene delivery vehicles are known in the art and include both viral and non-viral (e.g., naked DNA, plasmid) vectors. Viral vectors suitable for gene delivery are known to those skilled in the art. Such viral vectors include, for example, vectors derived from herpes viruses, baculovirus vectors, lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors (AAV), and Murine Stem Cell Virus (MSCV). Viral vectors may be replicative or non-replicative. Such vectors can be introduced into a number of suitable host cells using the methods disclosed or referenced herein, or other methods known to those skilled in the relevant art.

Non-viral vectors for gene delivery include naked DNA, plasmids, transposons, and mRNA, among others. Non-limiting examples include the pKK plasmid (Clonetech), pUC plasmid, pET plasmid (Novagen, madison, wisconsin), pRSET or pREP plasmid (Invitrogen, san diego, ca), pMAL plasmid (New England biological laboratories (New England Biolabs, beverly, massachusetts). Such vectors can be introduced into a number of suitable host cells using the methods disclosed or referenced herein, or other methods known to those skilled in the relevant art.

In certain embodiments, the vector comprises an Internal Ribosome Entry Site (IRES). In some embodiments, the vector includes a selectable marker, such as an ampicillin resistance gene (Amp). In some embodiments, the nucleic acid encodes a fluorescent protein, such as Green Fluorescent Protein (GFP). In some embodiments, the nucleic acid is suitable for subcloning into pMSCV-IRES-GFP between EcoRI and XhoI. In some embodiments, the vector contains a Multiple Cloning Site (MCS) for insertion of the desired gene.

Although the genetic code is degenerate in that most amino acids are represented by multiple codons (referred to as "synonymous" or "synonymous" codons), it is understood in the art that codon usage for a particular organism is nonrandom and biased towards a particular codon triplet. Accordingly, in some embodiments, the vector comprises a nucleotide sequence that has been optimized for expression in a particular type of host cell (e.g., by codon optimization). Codon optimization refers to the process in which a polynucleotide encoding a protein of interest is modified to replace a particular codon in the polynucleotide with a codon that encodes the same amino acid or amino acids but is more commonly used/recognized in a host cell expressing the nucleic acid. In some aspects, the polynucleotides described herein are codon optimized for expression in T cells.

Membrane-bound anti-cytokine constructs

Membrane-bound anti-cytokine constructs, examples of which are the mb-aIL6 construct of FIG. 1A, can be generated as described herein. The anti-cytokine construct may be specific for a variety of cytokines, such as IL-6, (TNF) - α, IL-1 β, IL-12, IL-17, IL-18, IFN γ, and the like.

Figure 1A shows a specific construct, which is an anti-IL 6 single chain variable fragment (anti-IL 6scFv) coupled to a hinge and transmembrane domain. The anti-IL 6scFv includes an anti-IL-6 variable light domain, an anti-IL-6 variable heavy domain, and a linker domain that links the variable light domain and the variable heavy domain. The relative positions of the variable light and heavy chain domains may be reversed, but they are both at the N' end of the transmembrane domain, shown in fig. 1A as the CD 8a hinge and transmembrane domain. The construct may also include an N-terminal signal peptide (not shown in fig. 1A), such as the CD 8a signal peptide. FIG. 1B is a schematic representation of the MSCV mb-aIL6-IRES-GFP plasmid.

A variety of linker domains are suitable. In some embodiments, the connector domain may be (G4S)_xWherein x is an integer from 1 to 100. In some embodiments, the connector domain may be (G4S)₃. In other embodiments, the linker domain may be one or more glycine residues. In other embodiments, the connector domain may be (EAAAK)₃。

A variety of hinge and transmembrane domains are suitable. In some embodiments, the hinge domain may be a CD 8a hinge domain. In some embodiments, the transmembrane domain may be a CD 8a transmembrane domain. In some embodiments, the hinge and transmembrane domain may be a CD 8a hinge and transmembrane domain. In some embodiments, the hinge can be a plurality of glycine and serine residues. In some embodiments, the transmembrane domain may be a transmembrane domain from CD4, CD8 β, CD16, CD28, CD32, CD34, CD64, CD137, FcRI γ, OX40, CD3 ζ, CD3, CD3 γ, CD3, TCR α, VEGFR2, FAS, or FGFR 2B.

Although the example of FIG. 1A is an anti-IL 6 construct, similar methods can be used to generate constructs for other cytokines, such as Tumor Necrosis Factor (TNF) - α, IL-1 β, IL-12, IL-17, IL-18, IFN γ, etc., and/or to block their receptors. For example, based on the schematic in FIG. 1A, the anti-IL 6scFv moiety can be replaced by a different scFv that specifically binds to a different cytokine, such as (TNF) - α (FIG. 11), IL-1 β, IL-12, IL-17, IL-18, or IFN γ. All the teachings herein are equally applicable to constructs expressing membrane bound proteins that neutralize cytokines.

Multiple neutralizing receptors may be expressed on the same cell or in different cell subsets to exert a comprehensive and long-lasting anti-inflammatory effect.

Method for producing transgenic host cells

Described herein are methods of making transgenic host cells (e.g., transgenic T cells). A transgenic host cell can be prepared, for example, by introducing one or more of the vector embodiments described herein into a host cell.

In one embodiment, the method comprises introducing into a host cell a vector comprising a nucleic acid encoding a membrane-bound single-chain variable fragment (scFv) of anti-IL 6(mb-aIL 6). In some embodiments, the nucleic acid of the vector can further encode a Chimeric Antigen Receptor (CAR), such as anti-CD 19-41BB-CD3 ζ. In some embodiments, the nucleic acid (e.g., a bicistronic vector) expresses mb-aIL6 and a CAR. In some embodiments, two separate vectors may be used to generate transgenic cells, such as transgenic T cells, that express mb-aIL6 and CAR.

In some embodiments, the one or more nucleic acids are integrated into the genome of the host cell. In some embodiments, the nucleic acid to be integrated into the host genome can be introduced into the host cell using any of a variety of suitable methods known in the art, including, for example, homologous recombination, CRISPR-based systems (e.g., CRISPR/Cas 9; CRISPR/Cpf1), and TALEN systems.

Value and range

Unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values expressed as ranges in different embodiments can take any specific value or subrange within the stated range, unless the context clearly dictates otherwise. "about" in reference to a numerical value generally refers to a range of values that fall within ± 8%, in some embodiments ± 6%, in some embodiments ± 4%, in some embodiments ± 2%, in some embodiments ± 1%, in some embodiments ± 0.5%, unless otherwise indicated or otherwise evident from the context.

Example embodiment: carrier

One example is a vector comprising a nucleic acid encoding a membrane-bound anti-IL 6(mb-aIL6) single-chain variable fragment (scFv). The mb-aIL 6scFv includes a) an anti-IL 6 single chain variable fragment (anti-IL 6scFv) comprising an anti-IL-6 variable light domain, an anti-IL-6 variable heavy domain, and a linker domain connecting the variable light domain and the variable heavy domain; and b) a hinge and transmembrane domain coupled to the anti-IL 6 scFv.

In some embodiments, one or more of the anti-IL-6 variable light chain domain and the anti-IL-6 variable heavy chain domain are human anti-IL 6 variable light chain domain and variable heavy chain domain. In some embodiments, the variable light chain domain has at least 90% sequence identity to SEQ ID No. 4. In some embodiments, the variable heavy chain domain has at least 90% sequence identity to SEQ ID No. 8.

In some embodiments, the connector domain is (G4S)_xWherein x is an integer from 1 to 100. In some embodiments, the connector domain is (G4S)₃. In some embodiments, the linker domain is one or more glycine residues. In some embodiments, the connector domain is (EAAAK)₃。

In some embodiments, the hinge and transmembrane domain is a CD 8a hinge and transmembrane domain. In some embodiments, the hinge comprises a plurality of glycine and serine residues. In some embodiments, the transmembrane domain is a transmembrane domain from CD4, CD8 β, CD16, CD28, CD32, CD34, CD64, CD137, FcRI γ, OX40, CD3 ζ, CD3, CD3 γ, CD3, TCR α, VEGFR2, FAS, or FGFR 2B.

In some embodiments, the nucleic acid further encodes a Chimeric Antigen Receptor (CAR), such as an anti-CD 19-41BB-CD3 ζ Chimeric Antigen Receptor (CAR). In some embodiments, mb-aIL6 is coupled to anti-CD 19-41BB-CD3 ζ via P2A sequence.

Example embodiment: carrier

Another example is a vector comprising a nucleic acid encoding a single chain variable fragment (scFv) of anti-IL 6(aIL 6). The aIL 6scFv comprises: a) an anti-IL 6 single-chain variable fragment (anti-IL 6scFv) comprising an anti-IL-6 variable light domain, an anti-IL-6 variable heavy domain, and a linker domain connecting the variable light domain and the variable heavy domain.

Example embodiment: carrier

Another embodiment is a vector comprising a nucleic acid encoding an anti-IL 6 Chimeric Antigen Receptor (CAR). The anti-IL 6CAR comprises: a) an anti-IL 6 single-chain variable fragment (anti-IL 6scFv) comprising an anti-IL-6 variable light domain, an anti-IL-6 variable heavy domain, and a first linker domain that links the variable light domain and the variable heavy domain; b) a second linker domain at the N-terminus of the anti-IL 6 scFv; c) a hinge and transmembrane domain at the N-terminus of the second linker domain; and d) an intracellular signaling domain at the N-terminus of the hinge and transmembrane domains.

In some embodiments, the intracellular signaling domain is a 41BB domain.

In some embodiments, the vector further comprises a co-stimulatory domain at the N-terminus of the intracellular signaling domain. In some embodiments, the costimulatory domain is the CD3 zeta domain.

Example embodiment: carrier

Another embodiment is a vector comprising a nucleic acid encoding a membrane-bound IL-6 receptor. The membrane-bound IL-6 receptor includes: a) an extracellular domain; b) a linker domain at the N-terminus of the extracellular domain; c) an IL-6 receptor alpha domain; and d) a transmembrane domain.

In some embodiments, the extracellular domain is a gp130 extracellular domain.

In some embodiments, the transmembrane domain is a CD 8a transmembrane domain.

Example embodiment: mammalian T cells

Another embodiment is according to any of the embodiments described herein, comprising a transgenic mammalian T cell encoding a membrane-bound anti-IL 6(mb-aIL6) single-chain variable fragment (scFv).

In some embodiments, the mb-aIL 6scFv may comprise: a) an anti-IL 6 single-chain variable fragment (anti-IL 6scFv) comprising an anti-IL-6 variable light domain, an anti-IL-6 variable heavy domain, and a linker domain connecting the variable light domain and the variable heavy domain; and b) a hinge and transmembrane domain coupled to the anti-IL 6 scFv.

In some embodiments, the mammalian T cell is a human T cell. In some embodiments, the mammalian T cells are human peripheral blood T lymphocytes.

Example embodiment: method of inhibiting proliferation of IL-6 dependent cells in a mammal

Another embodiment is a method of inhibiting the proliferation of IL-6-dependent cells in a mammal. The method comprises the following steps: membrane-bound anti-IL 6(mb-aIL6) single-chain variable fragment (scFv) was expressed in T cells. The mb-aIL 6scFv may be according to any of the examples described herein.

In some embodiments, the mb-aIL 6scFv comprises: a) an anti-IL 6 single-chain variable fragment (anti-IL 6scFv) comprising an anti-IL-6 variable light domain, an anti-IL-6 variable heavy domain, and a linker domain connecting the variable light domain and the variable heavy domain; and b) a hinge and transmembrane domain coupled to the anti-IL 6 scFv.

In some embodiments, the mammal is a human.

Example embodiment: methods of reducing IL-6 concentration in mammals

Another embodiment is a method of reducing IL-6 concentration in a mammal. The method comprises the following steps: membrane-bound anti-IL 6(mb-aIL6) single-chain variable fragment (scFv) was expressed in T cells, and the T cells were contacted with a liquid containing IL-6. The mb-aIL 6scFv may be according to any of the examples described herein.

In some embodiments, the mb-aIL6 includes: a) an anti-IL 6 single-chain variable fragment (anti-IL 6scFv) comprising an anti-IL-6 variable light domain, an anti-IL-6 variable heavy domain, and a linker domain connecting the variable light domain and the variable heavy domain; and b) a hinge and transmembrane domain coupled to the anti-IL 6 scFv.

In some embodiments, the mammal is a human.

In some embodiments, the method further comprises culturing the T cell to generate a new T cell that expresses the mb-aIL 6.

In some embodiments, the method further comprises reducing the risk of Cytokine Release Syndrome (CRS), and wherein the T cell of the mammal expresses a chimeric antigen receptor.

In some embodiments, the chimeric antigen receptor is an anti-CD 19-41BB-CD3 ζ CAR.

In some embodiments, the chimeric antigen receptor comprises an anti-CD 22 domain, an anti-CD 20 domain, an anti-CD 123 domain, a B Cell Maturation Antigen (BCMA) domain, an anti-mesothelin domain, an anti-CD 7 domain, an anti-CD 2 domain, an anti-CD 5 domain, an anti-CD 3 domain, an anti-Lewis Y domain, an anti-EpCam domain, an anti-Her 2 domain, or an anti-Prostate Specific Membrane Antigen (PSMA).

In some embodiments, the CAR further comprises a 4-1BB domain, a CD3 zeta domain, a CD28 domain, an inducible T cell costimulatory factor (ICOS) domain, a DNAX activating protein 10(DAP10) domain, or a DNAX activating protein 12(DAP12) domain.

In some embodiments, the mammal has an autoimmune disease, and reducing the concentration of IL-6 in the mammal treats the autoimmune disease.

In some embodiments, the mammal has rheumatoid arthritis, and decreasing the concentration of IL-6 treats rheumatoid arthritis.

In some embodiments, the mammal has systemic lupus erythematosus, and decreasing the concentration of IL-6 treats systemic lupus.

In some embodiments, the mammal has an inflammatory disease, and reducing the concentration of IL-6 in the mammal treats the inflammatory disease.

In some embodiments, the mammal has graft-versus-host disease, and decreasing the concentration of IL-6 treats graft-versus-host disease.

In some embodiments, the mammal has a lymphoproliferative disorder, and decreasing the concentration of IL-6 treats the lymphoproliferative disorder.

In some embodiments, the mammal has castleman's disease and reducing the concentration of IL-6 treats castleman's disease.

Example embodiment: carrier

In another embodiment, the vector comprises a nucleic acid encoding a membrane-bound anti-cytokine single-chain variable fragment (scFv). The membrane-bound anti-cytokine scFv can be according to any of the examples described herein.

In some embodiments, the membrane-bound anti-cytokine comprises: a) an anti-cytokine single chain variable fragment (anti-cytokine scFv) comprising an anti-cytokine variable light chain domain, an anti-cytokine variable heavy chain domain, and a linker domain connecting the variable light chain domain and the variable heavy chain domain; and b) a hinge and transmembrane domain coupled to the anti-cytokine scFv.

In some embodiments, the anti-cytokine is anti (TNF) -alpha, anti IL-1 beta, anti IL-12, anti IL-17, anti IL-18, or anti IFN gamma.

Examples of the use of

Materials and methods

Cells

Human cell lines Nalm-6(B cell acute lymphoblastic leukemia, ALL), Jurkat (T cell ALL), THP-1 and U937 (acute monocytic leukemia), DS-1(B cell lymphoma), and HEK293T (embryonic kidney fibroblast) were purchased from American type culture Collection (Manassas, Va.). The B-ALL cell line OP-1 was developed in our laboratory.¹¹Nalm-6, OP-1, THP-1, U937 and Jurkat cells were maintained supplemented with 10% fetal bovine bloodQing (FBS) (Hyclone GE Healthcare, Rogen, Utah) and 1% penicillin/streptavidin (P/S) (PAN-Biotech, Edinba, Germany) in RPMI-1640 (Sermer Feishell Scientific, Waltham, Mass.). DS-1 cells were maintained in RPMI-1640 (Seimer Feishell technology Co.) supplemented with 10% FBS, 1% P/S and 1ng/mL interleukin-6 (IL-6). HEK-293T was maintained in DMEM (Hyclone Laboratories) supplemented with 10% FBS and 1% P/S.

DS-1 and Nalm-6 were transduced with Murine Stem Cell Virus (MSCV) -Internal Ribosome Entry Site (IRES) -Green Fluorescent Protein (GFP) retroviral vectors containing the firefly luciferase gene (st. jude childhood Research Vector Development and Production Shared Resource, obtained from the institute of santa fursiensis, tennessee), and selected for GFP expression using a MoFlo cell sorter (Beckman Coulter, brayama, california). DS-1 and OP-1 were transduced with mCSV-IRES-GFP retroviral vectors containing mCherry and selected for mCherry expression using a MoFlo cell sorter.

To induce differentiation of THP-1, 2X10 was added⁶THP-1 cells were cultured for 72 hours in 10mL RPMI-1640 supplemented with 10% FBS, 1% P/S and 20ng/mL phorbol 12-myristate 13-acetate (PMA). Differentiated THP-1 cells were then harvested using 1% EDTA (Merck, Kennerworth, N.J.).

Peripheral Blood mononuclear cells were separated by density gradient from the waste anonymous by-product of platelet donations provided by the National University Hospital Blood Donation center (National University Hospital Blood Donation center). Monocytes were cultured with Dynabeads human T activator CD3/CD28 (ThermoFisher) in RPMI-1640 supplemented with 10% FBS, 1% P/S, and 120IU/ml interleukin-2 (IL-2) (Novartis, Basel, Switzerland) for 3 days. On day 4, the anti-CD 3/CD28 beads were removed and the cells were re-stimulated with fresh anti-CD 3/CD28 beads. The expanded T cells were then cultured in RPMI-1640 supplemented with 10% FBS, 1% P/S and 120IU/ml IL-2.

Plasmid and retroviral transduction

The heavy and light chain domains of the anti-IL 6scFv in mb-aIL6 were derived from the published sequence of the human anti-IL 6 monoclonal antibody AME-19a, and matched with 15 amino acids [ (G4S)₃]Linker ligation to form a single chain variable fragment (scFv); this construct was synthesized by the national institute of gold (GenScript) (south kyo, china). The scFv was linked to the CD8 α hinge and transmembrane domain ("mb-aIL 6"). The anti-CD 19-41BB-CD3 ζ construct was previously developed in our laboratory.¹²The P2A sequence for linking mb-aIL6 and anti-CD 19-41BB-CD3 ζ was previously reported.¹³All constructs were subcloned into pMSCV-IRES-GFP between EcoRI and XhoI.

Preparation of retroviral supernatants and transduction were performed as described previously.¹⁴Briefly, T cells were incubated with retroviral supernatants at 37 ℃ in the presence of RetroNectin (Takara, Otsumadin, Japan). The retroviral supernatant was replaced every 12 hours thereafter with fresh harvested supernatant for the next three days. Transduced T cells were subsequently harvested and cultured in RPMI-1640 supplemented with 10% FBS, 1% P/S and 200IU/mL IL-2.

Surface staining of transduced cells

Surface expression of mb-aIL6 and anti-CD 19-41BB-CD3 ζ were examined by flow cytometry. For mb-aIL6, secondary staining was performed using biotin-conjugated goat anti-human f (ab) 2 (Jackson ImmunoResearch, west grove, pa) followed by Allophycocyanin (APC) -conjugated streptavidin (BD biosciences, san jose, ca). Cells were also labeled with human IL-6 conjugated to biotin (ebola, cambridge, uk), followed by streptavidin APC; soybean trypsin inhibitor conjugated to biotin (from R & D, minneapolis, mn) was used as a negative control. CD19-myc is a soluble fusion protein produced by our laboratory that contains the extracellular domain of human CD19 linked to a myc-tag for the specific detection of anti-CD 19-41BB-CD3 ζ. T cells were incubated with CD19-myc for 30 minutes, followed by anti-myc (cell signaling technology, denfoss, ma) conjugated with R-Phycoerythrin (PE). For the cellular immunophenotype, T cells were labeled with anti-CD 3 APC, anti-CD 56 PE, anti-CD 4V 450, and anti-CD 8 PerCP (BD biosciences). Cell staining was analyzed using an Accuri C6 or Fortessa flow cytometer (BD biosciences).

IL-6 depletion assay

To measure IL-6 depletion, 2X10 was used⁶Each T cell was cultured in 1mL of RPMI-1640 containing 1ng of IL-6 for 2 hours. In another experiment, 0.5-2X10 was run⁶Each T cell was cultured in 1mL of RPMI-1640 containing 10IU of IL-6 for 2 hours. In yet another experiment, 2x10 was set⁶Individual T cells were cultured in 1mL RPMI-1640 containing 1ng IL-6 for different time intervals ranging from 20 minutes to 2 hours. In further experiments, 0.5x10 was set⁶The cells were cultured in 1mL RPMI containing 25-200pg/mL recombinant human IL-6 at 37 ℃ for 2 hours. In another further experiment, 0.2x10 was set⁶Each T cell was cultured in 1mL of RPMI-1640 containing 1ng of IL-6 for 2-72 hours. At the end of the incubation, the supernatant was harvested, filtered with a 0.22 μm filter and diluted 1: 10. The level of IL-6 was measured by ELISA using a human IL-6Platinum ELISA kit (Seimer Feishell Co.). The concentration of IL-6 in each sample was determined using interpolation from the calculated standard curve.

For Stat3 phosphorylation measurements, 2x10⁶Each cell was seeded in 1mL RPMI-1640 containing 10IU IL-6 for 2 hours. The supernatant was harvested and mixed with 0.2x10⁶U937 cells at 37 degrees C were incubated for 15 minutes. Lysis fixation buffer (Lysefix buffer) (BD biosciences) was added, and the sample was further maintained at 37 ℃ for 10 minutes. Cells were then washed and placed in Perm III buffer on ice for 30 minutes. After three washes, PE-conjugated anti-Stat 3(pY705) antibody (BD biosciences) was added. After 1 hour, the cells were washed and analyzed by flow cytometry.

To determine the effect of mb-aIL6 depletion of IL-6 on the growth of the IL-6 dependent cell line DS-1, the following was performed1.5x10⁴T cells were incubated with DS-1 cells transduced with mCherry (at a ratio of 1: 1) in RPMI-1640 supplemented with 10% FBS, 1% P/S and 0.5ng/ml IL-6. For expanded T cells, 120IU/ml IL-2 was added. DS-1 cells were starved for 72 hours prior to the start of the experiment. IL-2 and IL-6 were added every 2 days during the culture. 4x images of each well were captured every 4 hours using the IncuCyte live cell analysis system (Essen Biosciences, ann arba, michigan). Cell counts were measured using fluorescence and total red object integrated intensity (total red object integrated intensity) was used as a measure of the amount of DS-1mCherry in the wells. In other experiments, T cells, OP-1 and differentiated THP-1 were similarly cultured at a ratio of 1:5: 1. Differentiated THP-1 was inoculated 1 hour before the start of the assay. The IncuCyte system was used as above to capture a 4x image of each well. After 40 hours, supernatants from each well were harvested, diluted 1:5, and IL-6 measured by ELISA as described above.

Interferon-gamma (IFN γ) production, CD107a expression, cytotoxicity and proliferation assays

To test for IFN γ production, 1x10 was used⁵Individual T cells were cultured with OP-1 cells at a ratio of 1: 1. After 1 hour, GolgiPlug (BD biosciences) was added to the cells and cultured for an additional 5 hours. After permeabilization of the cell membrane with permeabilizing reagent 8E developed in our laboratory, the cells were labeled with PE-conjugated anti-human IFN γ antibody (BD biosciences) and analyzed by flow cytometry.

To measure exocytosis of cytotoxic particles, cells were cultured as described above. At the beginning of the culture, PE-conjugated anti-human CD107a antibody (BD biosciences) was added. After 1 hour, GolgiStop (BD biosciences) was added and the culture was continued for 3 hours, and then analyzed by flow cytometry.

To test for cytotoxicity, OP-1 cells were labeled with calcein-AM red (invitrogen, carlsbad, ca) and plated into 96-well round bottom plates. T cells were added at a 1: 1E: T ratio (1X 10)⁵) And co-cultured for 4 hours. As described previously, live target cells (calcein-AM positive) were treated by flow cytometryThe lines are counted.¹⁴

To measure cell proliferation, 1 × 10 was used⁵T cells were co-cultured with irradiated OP-1 at an E: T ratio of 1:1 in RPMI-1640 supplemented with 10% FBS, 1% P/S and 120IU/ml IL-2. IL-2 was added to each well every 2 days. On days 7, 14 and 21, T cells were counted by flow cytometry. After cell counting, freshly irradiated OP-1 cells were added to reconstitute the E: T ratio of 1: 1.

Xenograft experiments

Cg-Prkdc in NOD^scid IL2rg^tm1WjlIntra-peritoneal injection (i.p.; 1X 10) in/SzJ (NOD/scid IL2RGnull) mice (Jackson laboratory)⁶Individual cells/mouse) luciferase-expressing DS-1 cells. Two days after DS-1 inoculation, mice received ip injections of 1X10 transduced with GFP only⁷T cells, 1X10 transduced with MSCV-mb-aIL6⁷T cells, or RPMI 1640 with 10% FBS (instead of T cells). All mice received 20000IU IL-2 and 1000IU IL-6 every 2 days i.p. Tumor burden was measured twice weekly using the Xenogen IVIS-200 system (caliper life science, waltham, ma) after i.p. injection of d-luciferin potassium salt in water (Perkin Elmer, waltham, ma) (2 mg/mouse). Luminescence was measured with live imaging 3.0software (Living Image 3.0 software).

Luciferase-expressing Nalm-6 cells were injected intravenously in NOD/scid IL2RGnull mice (i.v.; FIGS. 10A-B0.5X 10)⁶Individual cells/mouse, FIGS. 10C-F are 1X10⁶Individual cells/mouse). Three days later, mice received i.v. injection of 2x10 transduced with anti-CD 19-41BB-CD3 zeta CAR⁷Individual T cells, 2x10 transduced with constructs containing CAR and mb-aIL6 ("DUAL⁷T cells, or RPMI 1640 with 10% FBS (instead of T cells). All mice received 20000IU IL-2 intraperitoneally every 2 days. Tumor burden was measured twice weekly using Xenogen IVIS-200 system (caliper life sciences, waltham, ma) after i.p. injection of d-luciferin potassium salt in water (2 mg/mouse). Luminescence was analyzed with in vivo imaging 3.0 software.

Results

Design, expression and specificity of mb-aIL6

To generate the membrane-bound anti-IL 6 construct, single chain variable fragments (scFv) were synthesized from the sequences of the variable light and heavy chains of the human anti-IL-6 antibody AME-19a and linked to the CD 8a hinge and transmembrane domains (fig. 1A). The construct was placed in a MSCV retroviral vector containing an IRES and GFP (FIG. 1B). This retroviral vector was used to transduce Jurkat T cells. GFP expression was higher in cells transduced with MSCV-mb-aIL 6: 98% of the cells were GFP positive. To detect mb-aIL6 on the surface of transduced Jurkat cells, cells were labeled with biotin-conjugated goat anti-human f (ab)' antibody followed by streptavidin APC. As shown in FIG. 1C, mb-aIL6 was detected in essentially all GFP-expressing Jurkat cells, whereas cells transduced with vectors containing GFP only ("mock") were mb-aIL6 negative.

To determine whether mb-aIL6 expressed on the cell surface was able to bind human IL-6, transduced Jurkat cells were exposed to biotin-conjugated human IL-6 for 10 minutes; cells were then labeled with streptavidin APC. As shown in FIG. 1D, mb-aIL6-Jurkat cells bound IL-6 at a level proportional to GFP levels, and thus the receptor expressed: 99% of the cells bound IL-6, whereas cells labeled with biotinylated control protein (soybean trypsin inhibitor) remained unstained.

Neutralization of IL-6 with mb-aIL6 cells

Binding of mb-aIL6 to IL-6 was confirmed by the following experiment: in these experiments, Jurkat cells were cultured in medium containing recombinant human IL-6(1ng/mL) for 2 hours; residual IL-6 recovered after incubation in the supernatant was measured by ELISA. After 2 hours of culture, the concentration of IL-6 in the supernatant from mb-aIL6 Jurkat cells was 0.163ng/mL compared to the concentration of IL-6 in the supernatant from mock-transduced Jurkat cells, which was 0.941ng/mL (FIG. 2A).

To determine the number of mb-aIL6 Jurkat cells required to neutralize IL-6, from 0.25X10 was used⁶One cell/mL to 2X10⁶Increased concentration of individual cells/mL of cells. Removal of IL-6 from the supernatant was cell dose dependent (FIG. 2B). In parallel withIn the assay, the kinetics of IL-6 removal from mb-aIL6 cells was measured. As shown in fig. 2C, IL-6 neutralization was time-dependent, with nearly 90% neutralized after 30 minutes and became undetectable after 120 minutes. Notably, this curve is related to a first order exponential decay curve (R)²0.9957) showed a half-life of 7.443 minutes for IL-6 and a K of 0.09313.

To test whether mb-aIL 6-expressing Jurkat cells could also neutralize low concentrations of IL-6, IL-6 depletion assays were established using IL-6 concentrations ranging from 0.025 to 0.2 ng/mL. As shown in FIG. 2D, Jurkat cells expressing mb-aIL6 also neutralized most of the IL-6. Consistent with previous experiments, IL-6 levels at various concentrations were reduced by 3.8-5.4 fold in mb-aIL6 expressing Jurkat cells compared to mock-transduced cells.

We speculate that cell proliferation will generate new mb-aIL6 cells that will continue to neutralize IL-6 in prolonged cell culture. To test this concept, we used previously determined culture conditions (FIG. 2B), which were insufficient to neutralize IL-6 within 2 hours, and continued the culture for 72 hours. At 24 hours, most of the IL-6 had been removed from the supernatant (FIG. 2E).

Neutralization of the functional consequences of IL-6 with mb-aIL 6T cells

U937 is can be IL-6 stimulated monocyte cell line.^15,16Upon binding to the IL-6 receptor, IL-6 triggers Stat3 phosphorylation.¹⁷IL-6 containing supernatants were tested for their ability to elicit Stat3 phosphorylation in U937 after exposure to mb-aIL6 transduced or mock transduced Jurkat cells for 2 hours. Stat3 phosphorylation (P) was readily detected after 15 min exposure to supernatants containing 1ng/mL IL-6<0.001; n is 3; fig. 3A). Similar phosphorylation levels (P ═ insignificant) were observed if IL-6 supernatants had been collected from cultures containing mock-transduced Jurkat cells. In contrast, when the culture contained mb-aIL6 Jurkat cells, the Stat3 phosphorylation level was significantly lower than the Stat3 phosphorylation level measured in U937 cells, which were exposed to the supernatant of IL-6 or IL-6-containing mimetic transduced Jurkat cells (P937 cells)<0.001 for either comparison) and compared to Stat3 phosphorylated water of unstimulated cellsFlat (P ═ not significant).

DS-1 is a B lymphoma cell line that requires IL-6 for its proliferation.¹⁸We determined whether co-culture with mb-aIL 6-expressing Jurkat cells affected DS-1 amplification. For this purpose, continuous live cell imaging recordings of DS-1 cells transduced with a 5 day mCherry were used. As shown in FIG. 3B, DS-1 cultured in medium containing IL-6 rapidly expanded in the presence of mock-transduced Jurkat, while expansion was significantly reduced if mb-aIL6 transduced Jurkat cells were present in the culture. Under these conditions, the growth rate of DS-1 was similar to that observed in cultures lacking IL-6, regardless of the presence of mock-transduced or mb-aIL6 transduced Jurkat cells. Taken together, these results demonstrate the ability of mb-aIL 6-expressing cells to neutralize the effects of IL-6.

Expression of mb-aIL6 in human peripheral blood T lymphocytes

In the next set of experiments, it was determined whether mb-aIL-6 could be expressed on the surface of peripheral blood T lymphocytes. For this purpose, peripheral blood mononuclear cells were stimulated with anti-CD 3/CD28 beads and then transduced with MSCV-mb-aIL6 retroviral vectors. Cells transduced with vectors containing GFP only ("mock") were used as controls. As shown in FIGS. 4A and 4B, mb-aIL6 was highly expressed on the surface of transduced GFP + T lymphocytes and efficiently bound IL-6. The immunophenotype of mb-IL 6-expressing T cells remained essentially the same as that of mock-transduced T cells (fig. 4C).

It was tested whether mb-IL 6-expressing T cells could inhibit the growth of the IL-6 dependent cell line DS-1. FIG. 4D shows that DS-1 growth is significantly inhibited, indicating that mb-aIL6 expressed in T lymphocytes can neutralize IL-6 and Jurkat cells expressing this receptor.

Membrane-bound anti-IL-6 and anti-CD 19-41BB-CD3z CAR can be co-expressed in T lymphocytes

If CAR T cells express mb-aIL6 in addition to CAR, CRS development can be prevented by neutralizing IL-6 secreted by activated T lymphocytes and macrophages in the microenvironment.

As a first step in testing this concept, it was determined whether mb-aIL6 and CAR could be simultaneously testedAnd (4) effective expression. To this end, a bicistronic MSCV vector containing the genes encoding mb-aIL6 and anti-CD 19-41BB-CD3 ζ CAR was developed (FIG. 5A).¹²To specifically detect anti-CD 19 CARs, the extracellular domain of human CD19 molecule was linked to a myc tag and cells were stained with an anti-myc antibody. Figure 5B shows that both mb-aIL6 and anti-CD 19CAR can be expressed at high levels in peripheral blood T cells. Like mb-aIL6 expressed alone, mb-aIL6 expressed in conjunction with CAR efficiently neutralized IL-6 (fig. 5C).

Expression of mb-aIL6 did not affect CAR T cell function

It was determined whether expression of mb-aIL6 and IL-6 neutralization would affect T cell function activated by CAR. The production of IFN γ after co-culture with CD19+ target cell OP-1 was tested using T lymphocytes from 3 donors expressing mb-aIL6 or anti-CD 19CAR and T cells expressing both recipients. Regardless of whether mb-aIL6 was expressed, IFN γ production was high in CAR-expressing cells, while only mb-aIL 6-expressing T cells secreted IFN γ levels similar to those of mock-transduced cells (fig. 6A).

In the presence of CD19+ target cells, the CAR-expressing cells released cytotoxic particles as evidenced by staining with anti-CD 107a antibody. The percentage of CD107a + cells was similar regardless of mb-aIL6 expression, while cells expressing only mb-aIL6 remained CD107a negative (fig. 6B). Consistent with this result, the percent cytotoxicity against CD19+ target driven by CAR remained unchanged due to the presence of mb-aIL6 (fig. 6C).

An important functional characteristic of second and subsequent generation CARs is the ability to provide co-stimulation and maintain prolonged T cell proliferation.¹⁹As shown in figure 6D, anti-CD 19 CAR-T cells proliferated in the presence of CD19+ target cells for 4 weeks, and the proliferation rates in cells with and without mb-aIL6 were similar. In contrast, neither cells transduced with mb-aIL6 alone nor mock-transduced cells expanded, and expansion did not occur in the absence of target cells, regardless of CAR expression. Taken together, these results indicate that expression of mb-aIL6 does not affect CAR-driven T cell secretion of IFN γ, specific cytotoxicity, or cell proliferation.

The T cells expressing mb-aIL6 and CAR can kill target cells while neutralizing IL-6

During CRS triggered by CAR activation, IL-6 secreted by macrophages exacerbates its severity.^7-10

To mimic the interaction between CAR-T cells and macrophages, CAR-T cells were co-cultured with the monocyte cell line THP-1 (which secretes IL-6 in the presence of TNF-a and IFN γ);²⁰the latter cytokines are secreted by T lymphocytes upon activation together with IL-6.^9,21Thus, T lymphocytes transduced with mb-aIL6 and/or anti-CD 19CAR, THP-1 and CD19+ leukemia cells OP-1 were co-cultured for 40 hours (FIG. 7A). Killing of OP-1 cells and IL-6 levels in the supernatant were monitored. As shown in FIG. 7B, CAR T cells were effective at killing OP-1 regardless of mb-aIL6 expression or the presence or absence of THP-1. Notably, levels of IL-6 were significantly elevated in cultures containing both CAR-T and THP-1 cells, but these levels were essentially undetectable if CAR-T cells also expressed mb-aIL 6.

Xenograft experiments

To determine the ability of mb-aIL6 expressing T lymphocytes to neutralize IL-6 in vivo, experiments were performed with NOD/scid IL2RGnull mice implanted with luciferase-tagged DS-1 cells. Tumor growth was measured by in vivo imaging and compared between mice receiving T cells transduced with GFP alone and mice receiving T cells transduced with mb-aIL 6. Tumor burden was reduced in two of the three mice receiving mb-aIL6 transduced T cells, while tumor burden remained stable or increased in mice receiving mock-transduced T cells (fig. 9A-B).

Whether expression of mb-aIL6 affected the anti-tumor ability of anti-CD 19CAR T cells was tested. Experiments were performed with NOD/scid IL2RGnull mice implanted with luciferase-tagged Nalm-6 cells. Tumor growth was measured by in vivo imaging and compared between mice receiving T cells transduced with CAR only and mice receiving T cells transduced with a bicistronic construct containing CAR and mb-aIL 6. The CAR T cells had anti-leukemic activity regardless of whether mb-aIL6 was expressed (fig. 10A-F). Taken together, these results indicate that mb-aIL6 can neutralize IL-6 in vivo and does not significantly affect CAR function.

Discussion of the related Art

The results of this study indicate that mb-aIL6 carried by T lymphocytes is a powerful neutralizer for IL-6. Importantly, mb-aIL6 can be expressed on T lymphocytes in conjunction with CAR without affecting CAR efficacy. To this end, mb-aIL 6-expressing CAR-T cells should have a lower risk of triggering severe CRS while retaining their anti-tumor potential.

We have now devised additional receptors functionally related to mb-aIL6 but with unique characteristics (fig. 8A-C). Figure 8A is a schematic of a nucleic acid construct directed against a soluble form of the receptor (sec-aIL6) lacking the CD 8A transmembrane domain of mb-aIL6, which will be continuously secreted by the transduced cell and likely to spread more efficiently to the inflammatory microenvironment region. Figure 8B is a schematic of a nucleic acid construct in which mb-aIL6 is linked to a stimulatory domain and a co-stimulatory domain that are typically incorporated into a CAR (aIL 6-CAR). Cells bearing this receptor should proliferate after attachment of aIL6-CAR, amplifying IL-6 neutralization. FIG. 8C is a schematic representation of a nucleic acid construct in which scFv anti-IL 6 was replaced with an IL-6 receptor that lost signaling ability. This consists of the extracellular domain of gp130 fused to the IL-6 receptor alpha and the hinge plus transmembrane domain of CD8 alpha. A potential advantage of this format is that it may be less immunogenic than scFv-containing receptors.

Although this study focused on IL-6, similar approaches can be used to neutralize other proinflammatory cytokines, such as Tumor Necrosis Factor (TNF) - α (FIGS. 11A-B), IL-1 β, IL-12, IL-17, IL-18, IFN γ, etc., and/or block their receptors. For example, based on the schematic in FIG. 11A, the anti-IL 6scFv moiety can be replaced by a different scFv that specifically binds to a different cytokine, such as (TNF) - α (FIG. 11), IL-1 β, IL-12, IL-17, IL-18, or IFN γ. Multiple neutralizing receptors may be expressed on the same cell or in different cell subsets to exert a comprehensive and long-lasting anti-inflammatory effect.

Sequence of

mb-aIL6 sequence

CD 8a signal peptide nucleotide sequence (SEQ ID NO: 1):

CD8 α signal peptide amino acid sequence (SEQ ID NO: 2):

MALPVTALLLPLALLLHAARP

variable light chain nucleotide sequence of anti-IL 6 (SEQ ID NO: 3):

anti-IL-6 variable light chain amino acid sequence (SEQ ID NO: 4):

GSG linker nucleotide sequence (SEQ ID NO: 5):

GGCGGCGGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGATCC

GSG linker amino acid sequence (SEQ ID NO: 6): GGGGSGGGGSGGGGS

anti-IL-6 variable heavy chain nucleotide sequence (SEQ ID NO: 7):

anti-IL-6 variable heavy chain amino acid sequence (SEQ ID NO: 8):

CD 8a hinge and transmembrane domain nucleotide sequence (SEQ ID NO: 9):

CD 8a hinge and transmembrane domain amino acid sequence (SEQ ID NO: 10):

sec-aIL6

sec-aIL6 nucleotide sequence (SEQ ID NO: 11):

sec-aIL6 amino acid sequence (SEQ ID NO: 12):

aIL6BBz sequence

CD 8a signal peptide nucleotide sequence (SEQ ID NO: 13):

CD8 α signal peptide amino acid sequence (SEQ ID NO: 14): MALPVTALLLPLALLLHAARP

aIL 6scFv nucleotide sequence (SEQ ID NO: 15):

aIL 6scFv amino acid sequence (SEQ ID NO: 16):

CD 8a hinge and transmembrane domain nucleotide sequence (SEQ ID NO: 17):

CD 8a hinge and transmembrane domain amino acid sequence (SEQ ID NO: 18):

41BB domain nucleotide sequence (SEQ ID NO: 19):

41BB domain amino acid sequence (SEQ ID NO: 20):

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

CD3 zeta domain nucleotide sequence (SEQ ID NO: 21):

CD3 zeta domain amino acid sequence (SEQ ID NO: 22):

mb-gp130-IL6R sequence

gp130 domain nucleotide sequence (SEQ ID NO: 23):

gp130 domain amino acid sequence (SEQ ID NO: 24):

GSG linker nucleotide sequence (SEQ ID NO: 25):

GGAGGAGGAGGAAGCGGAGGAGGAGGCTCCGGCGGCGGCGGCTCT

GSG linker amino acid sequence (SEQ ID NO: 26): GGGGSGGGGSGGGGS

IL-6R domain nucleotide sequence (SEQ ID NO: 27):

IL-6R domain amino acid sequence (SEQ ID NO: 28):

CD 8a hinge and transmembrane domain nucleotide sequence (SEQ ID NO: 29):

CD 8a hinge and transmembrane domain amino acid sequence (SEQ ID NO: 30):

mb-aTNF alpha sequence

CD 8a signal peptide nucleotide sequence (SEQ ID NO: 31):

CD8 α signal peptide amino acid sequence (SEQ ID NO: 32):

MALPVTALLLPLALLLHAARP

anti-TNF α variable light chain nucleotide sequence (SEQ ID NO: 33):

anti-TNF α variable light chain amino acid sequence (SEQ ID NO: 34):

GSG linker nucleotide sequence (SEQ ID NO: 35):

GGAGGAGGAGGATCCGGAGGAGGAGGATCTGGCGGCGGCGGCAGC

GSG linker amino acid sequence (SEQ ID NO: 36): GGGGSGGGGSGGGGS

anti-TNF α variable heavy chain nucleotide sequence (SEQ ID NO: 37):

anti-TNF α variable heavy chain amino acid sequence (SEQ ID NO: 38):

CD 8a hinge and transmembrane domain nucleotide sequence (SEQ ID NO: 39):

CD 8a hinge and transmembrane domain amino acid sequence (SEQ ID NO: 40):

reference to the literature

1.Tanaka T,Kishimoto T.The biology and medical implications of interleukin-6.Cancer Immunol Res.2014；2(4):288-294.

2.Hunter CA,Jones SA.IL-6as a keystone cytokine in health and disease.Nat Immunol.2015；16(5):448-457.

3.McInnes IB,Schett G.Pathogenetic insights from the treatment of rheumatoid arthritis.Lancet.2017；389(10086):2328-2337.

4.Chen X,Das R,Komorowski R,et al.Blockade of interleukin-6signaling augments regulatory T-cell reconstitution and attenuates the severity of graft-versus-host disease.Blood.2009；114(4):891-900.

5.Tawara I,Koyama M,Liu C,et al.Interleukin-6modulates graft-versus-host responses after experimental allogeneic bone marrow transplantation.Clin Cancer Res.2011；17(1):77-88.

6.Roddy JV,Haverkos BM,McBride A,et al.Tocilizumab for steroid refractory acute graft-versus-host disease.Leuk Lymphoma.2016；57(1):81-85.

7.Lee DW,Gardner R,Porter DL,et al.Current concepts in the diagnosis and management of cytokine release syndrome.Blood.2014；124(2):188-195.

8.Davila ML,Riviere I,Wang X,et al.Efficacy and Toxicity Management of19-28z CAR T Cell Therapy in B Cell Acute Lymphoblastic Leukemia.Sci Transl Med.2014；6(224):224ra225.

9.Maude SL,Barrett D,Teachey DT,Grupp SA.Managing cytokine release syndrome associated with novel T cell-engaging therapies.Cancer J.2014；20(2):119-122.

10.Park JH,Geyer MB,Brentjens RJ.CD19-targeted CAR T-cell therapeutics for hematologic malignancies:interpreting clinical outcomes to date.Blood.2016；127(26):3312-3320.

11.Manabe A,Coustan-Smith E,Kumagai M,et al.Interleukin-4 induces programmed cell death(apoptosis)in cases of high-risk acute lymphoblastic leukemia.Blood.1994；83(7):1731-1737.

12.Imai C,Mihara K,Andreansky M,Nicholson IC,Pui CH,Campana D.Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia.Leukemia.2004；18:676-684.

13.Szymczak-Workman AL,Vignali KM,Vignali DA.Design and construction of 2A peptide-linked multicistronic vectors.Cold Spring Harb Protoc.2012；2012(2):199-204.

14.Kudo K,Imai C,Lorenzini P,et al.T lymphocytes expressing a CD16 signaling receptor exert antibody-dependent cancer cell killing.Cancer Res.2014；74(1):93-103.

15.Taga T,Kawanishi Y,Hardy RR,Hirano T,Kishimoto T.Receptors for B cell stimulatory factor 2.Quantitation,specificity,distribution,and regulation of their expression.J Exp Med.1987；166(4):967-981.

16.Onozaki K,Akiyama Y,Okano A,et al.Synergistic regulatory effects of interleukin 6 and interleukin 1 on the growth and differentiation of human and mouse myeloid leukemic cell lines.Cancer Res.1989；49(13):3602-3607.

17.Zhong Z,Wen Z,Darnell JE,Jr.Stat3:a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6.Science.1994；264(5155):95-98.

18.Bock GH,Long CA,Riley ML,et al.Characterization of a new IL-6-dependent human B-lymphoma cell line in long term culture.Cytokine.1993；5(5):480-489.

19.Campana D,Schwarz H,Imai C.4-1BB chimeric antigen receptors.Cancer J.2014；20(2):134-140.

20.Sanceau J,Wijdenes J,Revel M,Wietzerbin J.IL-6 and IL-6 receptor modulation by IFN-gamma and tumor necrosis factor-alpha in human monocytic cell line(THP-1).Priming effect of IFN-gamma.J Immunol.1991；147(8):2630-2637.

21.Slifka MK,Whitton JL.Activated and memory CD8+T cells can be distinguished by their cytokine profiles and phenotypic markers.J Immunol.2000；164(1):208-216.

Incorporated by reference; equivalent forms

The teachings of all patents, published applications, and references cited herein are incorporated by reference in their entirety.

While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of embodiments encompassed by the appended claims.

Sequence listing

<110> Singapore National University (National University of Singapore)

Tan, Hong Ji Adrian

Campana, Dario

<120> neutralization of human cytokines with membrane-bound anti-cytokine non-signaling adhesive expressed in immune cells

<130> 4459.1149-001

<141> 2020-09-25

<150> 62/651,311

<151> 2018-04-02

<160> 40

<170> SIPOSequenceListing 1.0

<210> 1

<211> 63

<212> DNA

<213> Artificial sequence

<220>

<223> mb-aIL6 CD8 alpha signal peptide nucleotide sequence

<400> 1

atggccctgc ccgtgaccgc tctgctgctg cccctggctc tgctgctgca tgctgctaga 60

ccc 63

<210> 2

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> mb-aIL6 CD8 alpha signal peptide amino acid sequence

<400> 2

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 3

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> mb-aIL6 variable light chain nucleotide sequence against IL6

<400> 3

gaaatcgtcc tgacccagtc ccctgccaca ctgtccctgt ctccaggaga gagggccacc 60

ctgagctgct ccgcctctat cagcgtgtcc tacatgtatt ggtaccagca gaagccagga 120

caggcaccta ggctgctgat ctacgacatg tctaacctgg caagcggcat ccccgcacgc 180

ttctctggaa gcggatccgg cacagacttt acactgacca tcagctccct ggagcctgag 240

gatttcgccg tgtactattg catgcagtgg tccggctatc catacacatt tggcggcggc 300

accaaggtgg agatcaag 318

<210> 4

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> mb-aIL6 variable light chain amino acid sequence for anti-IL-6

<400> 4

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ile Ser Val Ser Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Asp Met Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Met Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 5

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> mb-aIL6 GSG linker nucleotide sequence

<400> 5

ggcggcggcg gctctggagg aggaggaagc ggaggaggag gatcc 45

<210> 6

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> mb-aIL6 GSG linker amino acid sequence

<400> 6

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 7

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223> anti-IL-6 mb-aIL6 variable heavy chain nucleotide sequence

<400> 7

gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggctc cctgcggctg 60

tcttgtgccg ccagcggctt caccttttct ccattcgcca tgagctgggt gagacaggca 120

ccaggcaagg gcctggagtg ggtggccaag atctcccctg gcggctcttg gacatactat 180

tccgacacag tgaccggccg gtttaccatc tccagagata acgccaagaa cagcctgtat 240

ctgcagatga atagcctgcg ggccgaggac acagccgtgt actattgtgc cagacagctg 300

tggggctact atgccctgga tatctggggc cagggcacca cagtgaccgt gtctagc 357

<210> 8

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> anti-IL-6 mb-aIL6 variable heavy chain amino acid sequence

<400> 8

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Phe

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Lys Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val

50 55 60

Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Leu Trp Gly Tyr Tyr Ala Leu Asp Ile Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 9

<211> 213

<212> DNA

<213> Artificial sequence

<220>

<223> mb-aIL6 CD8 alpha hinge and transmembrane domain nucleotide sequences

<400> 9

aagcctacca caaccccagc acccaggccc cctacacctg caccaaccat cgccagccag 60

ccactgtccc tgaggcccga ggcatgcagg cctgcagcag gaggcgccgt gcacacccgc 120

ggcctggact tcgcctgtga tatctacatc tgggcacccc tggctggaac ctgcggagtc 180

ctgctgctgt cactggtcat taccctgtat tgc 213

<210> 10

<211> 71

<212> PRT

<213> Artificial sequence

<220>

<223> mb-aIL6 CD8 alpha hinge and transmembrane domain amino acid sequences

<400> 10

Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

1 5 10 15

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

20 25 30

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

35 40 45

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

50 55 60

Leu Val Ile Thr Leu Tyr Cys

65 70

<210> 11

<211> 783

<212> DNA

<213> Artificial sequence

<220>

<223> sec-aIL6 nucleotide sequence

<400> 11

atggccctgc ccgtgaccgc tctgctgctg cccctggctc tgctgctgca tgctgctaga 60

cccgaaatcg tcctgaccca gtcccctgcc acactgtccc tgtctccagg agagagggcc 120

accctgagct gctccgcctc tatcagcgtg tcctacatgt attggtacca gcagaagcca 180

ggacaggcac ctaggctgct gatctacgac atgtctaacc tggcaagcgg catccccgca 240

cgcttctctg gaagcggatc cggcacagac tttacactga ccatcagctc cctggagcct 300

gaggatttcg ccgtgtacta ttgcatgcag tggtccggct atccatacac atttggcggc 360

ggcaccaagg tggagatcaa gggcggcggc ggctctggag gaggaggaag cggaggagga 420

ggatccgagg tgcagctggt ggagagcggc ggcggcctgg tgcagcccgg cggctccctg 480

cggctgtctt gtgccgccag cggcttcacc ttttctccat tcgccatgag ctgggtgaga 540

caggcaccag gcaagggcct ggagtgggtg gccaagatct cccctggcgg ctcttggaca 600

tactattccg acacagtgac cggccggttt accatctcca gagataacgc caagaacagc 660

ctgtatctgc agatgaatag cctgcgggcc gaggacacag ccgtgtacta ttgtgccaga 720

cagctgtggg gctactatgc cctggatatc tggggccagg gcaccacagt gaccgtgtct 780

agc 783

<210> 12

<211> 261

<212> PRT

<213> Artificial sequence

<220>

<223> sec-aIL6 amino acid sequence

<400> 12

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ile

35 40 45

Ser Val Ser Tyr Met Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

50 55 60

Arg Leu Leu Ile Tyr Asp Met Ser Asn Leu Ala Ser Gly Ile Pro Ala

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

85 90 95

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Met Gln Trp Ser

100 105 110

Gly Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

130 135 140

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu

145 150 155 160

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Phe Ala Met

165 170 175

Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Lys

180 185 190

Ile Ser Pro Gly Gly Ser Trp Thr Tyr Tyr Ser Asp Thr Val Thr Gly

195 200 205

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln

210 215 220

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

225 230 235 240

Gln Leu Trp Gly Tyr Tyr Ala Leu Asp Ile Trp Gly Gln Gly Thr Thr

245 250 255

Val Thr Val Ser Ser

260

<210> 13

<211> 63

<212> DNA

<213> Artificial sequence

<220>

<223> aIL6BBz CD8 alpha signal peptide nucleotide sequence

<400> 13

atggccctgc ccgtgaccgc tctgctgctg cccctggctc tgctgctgca tgctgctaga 60

ccc 63

<210> 14

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> aIL6BBz CD8 alpha signal peptide amino acid sequence

<400> 14

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 15

<211> 720

<212> DNA

<213> Artificial sequence

<220>

<223> aIL6BBz aIL 6scFv nucleotide sequence

<400> 15

gaaatcgtcc tgacccagtc ccctgccaca ctgtccctgt ctccaggaga gagggccacc 60

ctgagctgct ccgcctctat cagcgtgtcc tacatgtatt ggtaccagca gaagccagga 120

caggcaccta ggctgctgat ctacgacatg tctaacctgg caagcggcat ccccgcacgc 180

ttctctggaa gcggatccgg cacagacttt acactgacca tcagctccct ggagcctgag 240

gatttcgccg tgtactattg catgcagtgg tccggctatc catacacatt tggcggcggc 300

accaaggtgg agatcaaggg cggcggcggc tctggaggag gaggaagcgg aggaggagga 360

tccgaggtgc agctggtgga gagcggcggc ggcctggtgc agcccggcgg ctccctgcgg 420

ctgtcttgtg ccgccagcgg cttcaccttt tctccattcg ccatgagctg ggtgagacag 480

gcaccaggca agggcctgga gtgggtggcc aagatctccc ctggcggctc ttggacatac 540

tattccgaca cagtgaccgg ccggtttacc atctccagag ataacgccaa gaacagcctg 600

tatctgcaga tgaatagcct gcgggccgag gacacagccg tgtactattg tgccagacag 660

ctgtggggct actatgccct ggatatctgg ggccagggca ccacagtgac cgtgtctagc 720

<210> 16

<211> 240

<212> PRT

<213> Artificial sequence

<220>

<223> aIL6BBz aIL 6scFv amino acid sequence

<400> 16

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ile Ser Val Ser Tyr Met

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Asp Met Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Met Gln Trp Ser Gly Tyr Pro Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser

115 120 125

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

130 135 140

Ala Ser Gly Phe Thr Phe Ser Pro Phe Ala Met Ser Trp Val Arg Gln

145 150 155 160

Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Lys Ile Ser Pro Gly Gly

165 170 175

Ser Trp Thr Tyr Tyr Ser Asp Thr Val Thr Gly Arg Phe Thr Ile Ser

180 185 190

Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg

195 200 205

Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gln Leu Trp Gly Tyr

210 215 220

Tyr Ala Leu Asp Ile Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

225 230 235 240

<210> 17

<211> 207

<212> DNA

<213> Artificial sequence

<220>

<223> aIL6BBz CD8 alpha hinge and transmembrane domain nucleotide sequences

<400> 17

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180

ctgtcactgg ttatcaccct ttactgc 207

<210> 18

<211> 69

<212> PRT

<213> Artificial sequence

<220>

<223> aIL6BBz CD8 alpha hinge and transmembrane domain amino acid sequences

<400> 18

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

50 55 60

Ile Thr Leu Tyr Cys

65

<210> 19

<211> 126

<212> DNA

<213> Artificial sequence

<220>

<223> aIL6BBz 41BB domain nucleotide sequence

<400> 19

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 20

<211> 42

<212> PRT

<213> Artificial sequence

<220>

<223> aIL6BBz 41BB domain amino acid sequence

<400> 20

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 21

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> aIL6BBz CD3 zeta domain nucleotide sequence

<400> 21

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 22

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> aIL6BBz CD3 zeta structure domain amino acid sequence

<400> 22

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 23

<211> 978

<212> DNA

<213> Artificial sequence

<220>

<223> mb-gp130-IL6R gp130 domain nucleotide sequence

<400> 23

atgctgacac tgcagacatg gctggtccag gcactgttta tctttctgac aaccgagtcc 60

acaggcgaac tgctggatcc ttgcgggtac atctctccag agagccccgt ggtgcagctg 120

cactccaact tcaccgccgt gtgcgtgctg aaggagaagt gtatggacta ctttcacgtg 180

aacgccaatt atatcgtgtg gaagacaaac cacttcacca tccctaagga gcagtacaca 240

atcatcaata gaaccgccag ctccgtgacc ttcaccgata tcgccagcct gaacatccag 300

ctgacatgca atatcctgac cttcggccag ctggagcaga acgtgtatgg catcaccatc 360

atctccggcc tgccccctga gaagccaaag aacctgtctt gcatcgtgaa tgagggcaag 420

aagatgaggt gtgagtggga ccggggcaga gagacacacc tggagacaaa tttcaccctg 480

aagtccgagt gggccaccca caagtttgcc gactgcaagg ccaagaggga tacacccacc 540

agctgtacag tggattactc caccgtgtat tttgtgaaca tcgaagtgtg ggtggaggcc 600

gagaatgccc tgggcaaggt gaccagcgac cacatcaact tcgatcccgt gtacaaggtg 660

aagcctaacc caccccacaa tctgtctgtg atcaatagcg aggagctgtc tagcatcctg 720

aagctgacat ggaccaaccc ctccatcaag tctgtgatca tcctgaagta caatatccag 780

tatagaacaa aggacgccag cacctggtcc cagatccctc cagaggatac agcctccacc 840

aggtcctctt ttacagtgca ggacctgaag cctttcaccg agtacgtgtt ccggatccgg 900

tgtatgaagg aggacggcaa gggctactgg tctgattgga gcgaggaggc ctccggcatc 960

acctatgagg acaggcca 978

<210> 24

<211> 326

<212> PRT

<213> Artificial sequence

<220>

<223> mb-gp130-IL6R gp130 domain amino acid sequence

<400> 24

Met Leu Thr Leu Gln Thr Trp Leu Val Gln Ala Leu Phe Ile Phe Leu

1 5 10 15

Thr Thr Glu Ser Thr Gly Glu Leu Leu Asp Pro Cys Gly Tyr Ile Ser

20 25 30

Pro Glu Ser Pro Val Val Gln Leu His Ser Asn Phe Thr Ala Val Cys

35 40 45

Val Leu Lys Glu Lys Cys Met Asp Tyr Phe His Val Asn Ala Asn Tyr

50 55 60

Ile Val Trp Lys Thr Asn His Phe Thr Ile Pro Lys Glu Gln Tyr Thr

65 70 75 80

Ile Ile Asn Arg Thr Ala Ser Ser Val Thr Phe Thr Asp Ile Ala Ser

85 90 95

Leu Asn Ile Gln Leu Thr Cys Asn Ile Leu Thr Phe Gly Gln Leu Glu

100 105 110

Gln Asn Val Tyr Gly Ile Thr Ile Ile Ser Gly Leu Pro Pro Glu Lys

115 120 125

Pro Lys Asn Leu Ser Cys Ile Val Asn Glu Gly Lys Lys Met Arg Cys

130 135 140

Glu Trp Asp Arg Gly Arg Glu Thr His Leu Glu Thr Asn Phe Thr Leu

145 150 155 160

Lys Ser Glu Trp Ala Thr His Lys Phe Ala Asp Cys Lys Ala Lys Arg

165 170 175

Asp Thr Pro Thr Ser Cys Thr Val Asp Tyr Ser Thr Val Tyr Phe Val

180 185 190

Asn Ile Glu Val Trp Val Glu Ala Glu Asn Ala Leu Gly Lys Val Thr

195 200 205

Ser Asp His Ile Asn Phe Asp Pro Val Tyr Lys Val Lys Pro Asn Pro

210 215 220

Pro His Asn Leu Ser Val Ile Asn Ser Glu Glu Leu Ser Ser Ile Leu

225 230 235 240

Lys Leu Thr Trp Thr Asn Pro Ser Ile Lys Ser Val Ile Ile Leu Lys

245 250 255

Tyr Asn Ile Gln Tyr Arg Thr Lys Asp Ala Ser Thr Trp Ser Gln Ile

260 265 270

Pro Pro Glu Asp Thr Ala Ser Thr Arg Ser Ser Phe Thr Val Gln Asp

275 280 285

Leu Lys Pro Phe Thr Glu Tyr Val Phe Arg Ile Arg Cys Met Lys Glu

290 295 300

Asp Gly Lys Gly Tyr Trp Ser Asp Trp Ser Glu Glu Ala Ser Gly Ile

305 310 315 320

Thr Tyr Glu Asp Arg Pro

325

<210> 25

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> mb-gp130-IL6R GSG linker nucleotide sequence

<400> 25

ggaggaggag gaagcggagg aggaggctcc ggcggcggcg gctct 45

<210> 26

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> mb-gp130-IL6R GSG linker amino acid sequence

<400> 26

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 27

<211> 663

<212> DNA

<213> Artificial sequence

<220>

<223> mb-gp130-IL6R IL-6R domain nucleotide sequence

<400> 27

gtggatgtgc cccctgagga gccccagctg tcttgcttca ggaagtcccc tctgtctaac 60

gtggtgtgcg agtggggacc tcgcagcacc ccatccctga ccacaaaggc cgtgctgctg 120

gtgcggaagt tccagaatag ccctgccgag gactttcagg agccatgcca gtactctcag 180

gagagccaga agttcagctg tcagctggca gtgccagagg gcgatagctc cttttatatc 240

gtgtccatgt gcgtggcctc tagcgtgggc tccaagttct ctaagacaca gacctttcag 300

ggctgtggca tcctgcagcc tgacccaccc gccaacatca cagtgaccgc cgtggcccgg 360

aatccaagat ggctgtctgt gacatggcag gatccccaca gctggaactc ctctttctac 420

cggctgagat ttgagctgag gtatcgcgcc gagcggagca agacctttac cacatggatg 480

gtgaaggacc tgcagcacca ctgcgtgatc cacgatgcat ggagcggcct gaggcacgtg 540

gtgcagctga gagcacagga ggagttcgga cagggagagt ggagcgagtg gtccccagag 600

gcaatgggaa caccatggac cgagagccgc tcccctccag cagagaatga ggtgagcaca 660

cca 663

<210> 28

<211> 221

<212> PRT

<213> Artificial sequence

<220>

<223> mb-gp130-IL6R IL-6R structural domain amino acid sequence

<400> 28

Val Asp Val Pro Pro Glu Glu Pro Gln Leu Ser Cys Phe Arg Lys Ser

1 5 10 15

Pro Leu Ser Asn Val Val Cys Glu Trp Gly Pro Arg Ser Thr Pro Ser

20 25 30

Leu Thr Thr Lys Ala Val Leu Leu Val Arg Lys Phe Gln Asn Ser Pro

35 40 45

Ala Glu Asp Phe Gln Glu Pro Cys Gln Tyr Ser Gln Glu Ser Gln Lys

50 55 60

Phe Ser Cys Gln Leu Ala Val Pro Glu Gly Asp Ser Ser Phe Tyr Ile

65 70 75 80

Val Ser Met Cys Val Ala Ser Ser Val Gly Ser Lys Phe Ser Lys Thr

85 90 95

Gln Thr Phe Gln Gly Cys Gly Ile Leu Gln Pro Asp Pro Pro Ala Asn

100 105 110

Ile Thr Val Thr Ala Val Ala Arg Asn Pro Arg Trp Leu Ser Val Thr

115 120 125

Trp Gln Asp Pro His Ser Trp Asn Ser Ser Phe Tyr Arg Leu Arg Phe

130 135 140

Glu Leu Arg Tyr Arg Ala Glu Arg Ser Lys Thr Phe Thr Thr Trp Met

145 150 155 160

Val Lys Asp Leu Gln His His Cys Val Ile His Asp Ala Trp Ser Gly

165 170 175

Leu Arg His Val Val Gln Leu Arg Ala Gln Glu Glu Phe Gly Gln Gly

180 185 190

Glu Trp Ser Glu Trp Ser Pro Glu Ala Met Gly Thr Pro Trp Thr Glu

195 200 205

Ser Arg Ser Pro Pro Ala Glu Asn Glu Val Ser Thr Pro

210 215 220

<210> 29

<211> 213

<212> DNA

<213> Artificial sequence

<220>

<223> mb-gp130-IL6R CD8 alpha hinge and transmembrane domain nucleotide sequences

<400> 29

aagccaacca caacccctgc accacggccc cctacaccag cacctaccat cgcatcccag 60

ccactgtctc tgaggcctga ggcatgcagg ccagcagcag gaggagcagt gcacacccgg 120

ggcctggact tcgcctgtga tatctacatc tgggccccac tggctggcac ttgcggggtc 180

ctgctgctgt ccctggtcat cactctgtat tgc 213

<210> 30

<211> 71

<212> PRT

<213> Artificial sequence

<220>

<223> mb-gp130-IL6R CD8 alpha hinge and transmembrane domain amino acid sequences

<400> 30

Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

1 5 10 15

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

20 25 30

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

35 40 45

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

50 55 60

Leu Val Ile Thr Leu Tyr Cys

65 70

<210> 31

<211> 63

<212> DNA

<213> Artificial sequence

<220>

<223> mb-aTNF alpha CD8 alpha signal peptide nucleotide sequence

<400> 31

atggccctgc ctgtgaccgc cctgctgctg cctctggccc tgctgctgca cgccgcccgc 60

ccc 63

<210> 32

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> mb-aTNF alpha CD8 alpha signal peptide amino acid sequence

<400> 32

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 33

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<223> mb-aTNF alpha variable light chain nucleotide sequence for anti-TNF alpha

<400> 33

gaaatcgtcc tgacccagtc ccccgccaca ctgtctctga gcccaggaga gagggccacc 60

ctgagctgca gagcctccca gtctgtgagc tcctacctgg cctggtatca gcagaagcca 120

ggacaggcac caaggctgct gatctacgac gcatccaaca gggcaacagg catccccgca 180

cgcttcagcg gatccggatc tggcagcggc accgacttta cactgaccat ctctagcctg 240

gagcctgagg atttcgccgt gtactattgc cagcagcgca gcaattggcc ccctttcaca 300

tttggcccag gcaccaaggt ggatatcaag 330

<210> 34

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> mb-aTNF alpha variable light chain amino acid sequence of anti-TNF alpha

<400> 34

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu

65 70 75 80

Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp

85 90 95

Pro Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105 110

<210> 35

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> mb-aTNF alpha GSG linker nucleotide sequence

<400> 35

ggaggaggag gatccggagg aggaggatct ggcggcggcg gcagc 45

<210> 36

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> mb-aTNF alpha GSG linker amino acid sequence

<400> 36

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 37

<211> 378

<212> DNA

<213> Artificial sequence

<220>

<223> anti-TNF alpha mb-aTNF alpha variable heavy chain nucleotide sequence

<400> 37

caggtgcagc tggtggagtc cggcggcggc gtggtgcagc caggcaggtc cctgaggctg 60

tcttgtgcag caagcggctt catcttttcc tcttacgcaa tgcactgggt gcggcaggca 120

cctggaaacg gcctggagtg ggtggccttc atgtcctacg acggctctaa taagaagtat 180

gccgattccg tgaagggccg gtttacaatc agcagagaca actccaagaa taccctgtat 240

ctgcagatga actctctgag ggccgaggac acagccgtgt actattgtgc ccgggataga 300

ggaatcgcag caggaggaaa ttactattac tatggcatgg acgtgtgggg ccagggcacc 360

acagtgaccg tgagctcc 378

<210> 38

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> anti-TNF alpha mb-aTNF alpha variable heavy chain amino acid sequence

<400> 38

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Val

35 40 45

Ala Phe Met Ser Tyr Asp Gly Ser Asn Lys Lys Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Gly Ile Ala Ala Gly Gly Asn Tyr Tyr Tyr Tyr Gly

100 105 110

Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 39

<211> 216

<212> DNA

<213> Artificial sequence

<220>

<223> mb-aTNF alpha CD8 alpha hinge and transmembrane domain nucleotide sequences

<400> 39

aagcctacca caacccctgc accacggcca ccaacaccag cacctaccat cgcctctcag 60

cctctgagcc tgaggccaga ggcatgcagg ccagcagcag gaggagcagt gcacaccaga 120

ggcctggact ttgcctgtga tatctacatc tgggcccctc tggctgggac ttgcggggtg 180

ctgctgctgt cactggtcat cacactgtat tgttga 216

<210> 40

<211> 71

<212> PRT

<213> Artificial sequence

<220>

<223> mb-aTNF alpha CD8 alpha hinge and transmembrane domain amino acid sequences

<400> 40

Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

1 5 10 15

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

20 25 30

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile

35 40 45

Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser

50 55 60

Leu Val Ile Thr Leu Tyr Cys

65 70

Claims (20)

1. A vector comprising a nucleic acid encoding a membrane-bound anti-IL 6(mb-aIL6) single-chain variable fragment (scFv), the mb-aIL 6scFv comprising:

a) an anti-IL 6 single-chain variable fragment (anti-IL 6scFv) comprising an anti-IL-6 variable light domain, an anti-IL-6 variable heavy domain, and a linker domain connecting the variable light domain and the variable heavy domain; and

b) a hinge and transmembrane domain coupled to the anti-IL 6 scFv.

2. The vector of claim 1, wherein one or more of the anti-IL-6 variable light chain domain and the anti-IL-6 variable heavy chain domain is a human anti-IL 6 variable light chain domain and variable heavy chain domain.

3. The vector of claim 2, wherein the variable light chain domain has at least 90% sequence identity to SEQ ID NO 4.

4. The vector of claim 2, wherein the variable heavy chain domain has at least 90% sequence identity to SEQ ID NO 8.

5. The vector of any one of claims 1-4 wherein the linker domain is (G4S)_xWherein x is an integer from 1 to 100.

6. The vector of any one of claims 1-4 wherein the linker domain is (G4S)₃。

7. The vector of any one of claims 1-4, wherein the hinge and transmembrane domain is a CD 8a hinge and transmembrane domain.

8. The vector of any one of claims 1-4, wherein the nucleic acid further encodes a Chimeric Antigen Receptor (CAR).

9. The vector of any one of claim 8, wherein the chimeric antigen receptor is an anti-CD 19-41BB-CD3 ζ Chimeric Antigen Receptor (CAR).

10. The vector of any one of claims 9, wherein the mb-aIL6 is coupled to the anti-CD 19-41BB-CD3 ζ via P2A sequence.

11. A vector comprising a nucleic acid encoding an anti-IL 6 Chimeric Antigen Receptor (CAR), the anti-IL 6CAR comprising:

a) an anti-IL 6 single-chain variable fragment (anti-IL 6scFv) comprising an anti-IL-6 variable light domain, an anti-IL-6 variable heavy domain, and a first linker domain that links the variable light domain and the variable heavy domain;

b) a second linker domain at the N-terminus of the anti-IL 6 scFv;

c) a hinge and transmembrane domain at the N-terminus of the second linker domain; and

d) an intracellular signaling domain at the N-terminus of the hinge and transmembrane domains.

12. The vector of claim 11, wherein the intracellular signaling domain is a 41BB domain.

13. The vector of claim 11, further comprising a co-stimulatory domain at the N-terminus of the intracellular signaling domain.

14. The vector of claim 13, wherein the co-stimulatory domain is the CD3 zeta domain.

15. A method of reducing the concentration of IL-6 in a mammal, the method comprising: expressing a membrane-bound anti-IL 6(mb-aIL6) single-chain variable fragment (scFv) in a T cell, and contacting the T cell with a liquid comprising IL-6, wherein the mb-aIL6 comprises:

a) an anti-IL 6 single-chain variable fragment (anti-IL 6scFv) comprising an anti-IL-6 variable light domain, an anti-IL-6 variable heavy domain, and a linker domain connecting the variable light domain and the variable heavy domain; and

b) a hinge and transmembrane domain coupled to the anti-IL 6 scFv.

16. The method of claim 15, wherein the mammal is a human.

17. The method of claim 15 or 16, further comprising culturing the T cell to generate a new T cell expressing the mb-aIL 6.

18. The method of claim 15 or 16, further comprising reducing the risk of Cytokine Release Syndrome (CRS), and wherein the T cell of the mammal expresses a chimeric antigen receptor.

19. The method of claim 15 or 16, wherein the mammal has an autoimmune disease, an inflammatory disease, or a lymphoproliferative disorder, and wherein reducing the concentration of IL-6 in the mammal treats the autoimmune disease, the inflammatory disease, or the lymphoproliferative disorder, respectively.

20. The method of claim 19, wherein the mammal has rheumatoid arthritis, systemic lupus erythematosus, graft-versus-host disease, or castleman disease, and wherein decreasing the concentration of IL-6 treats rheumatoid arthritis, systemic lupus erythematosus, graft-versus-host disease, or castleman disease, respectively.

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